The blood clotting process, as a mechanism of hemostasis or in the generation of a pathological condition involving thrombi, requires two cooperating pathways: (1) following activation, triggered by the enzyme thrombin, circulating platelets adhere to one another accompanied by the release of factors like thromboxane A2 and the subsequent formation of a plug created from the aggregated platelets, and (2) the activation of a cascade of proteolytic enzymes and cofactors, most of which are plasma glycoproteins synthesized in the liver, to produce a thrombus. Thrombi are composed mainly of an insoluble fibrin network, which entraps circulating blood cells, platelets, and plasma proteins to form a thrombus.
Thromboembolic disease, i.e., the pathological blockage of a blood vessel by a blood clot, is a significant cause of mortality and morbidity. Most spontaneously developing vascular obstructions are due to the formation of intravascular blood clots, or thrombi. Small fragments of a clot may also detach from the body of a clot and travel through the circulatory system to lodge in distant organs and initiate further clot formation. Myocardial infarction, occlusive stroke, deep venous thrombosis (DVT), and peripheral arterial disease are well-known consequences of thromboembolic phenomena.
Plasminogen activators are currently the favored agents employed in thrombolytic therapy, all of which convert plasminogen to plasmin and promote fibrinolysis by disrupting the fibrin matrix (Creager M. A. & Dzau V. J., Vascular Diseases of the Extremities, ppgs. 1398-1406 in Harrison's Principles of Internal Medicine, 14th ed., Fauci et al., editors, McGraw-Hill Co., New York, 1998; the contents of which is incorporated herein by reference in its entirety). The most widely used plasminogen activators include a recombinant form of tissue-type plasminogen activator (tPA), urokinase (UK) and streptokinase (SK), as well as a new generation of plasminogen activators selected for improved pharmacokinetics and fibrin-binding properties. All of these plasminogen activators, however, act indirectly to effect lysis and require an adequate supply of their common substrate, plasminogen, at the site of the thrombus.
UK and tPA convert plasminogen to plasmin by cleaving the Arg561-Va1562 peptide bond. The resulting two polypeptide chains of plasmin remain joined by two interchain disulfide bridges. The light chain of 25 kDa carries the catalytic center and is homologous to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five triple-loop kringle structures with highly similar amino acid sequences. Some of these kringles contain so-called lysine-binding sites that are responsible for plasminogen and plasmin interaction with fibrin, α2-antiplasmin, or other proteins. Variant forms of truncated plasmin, including variants lacking some or all of the kringle regions of the plasmin heavy chain, are disclosed by Wu et al. in U.S. Pat. No. 4,774,087, incorporated herein by reference in its entirety. SK and staphylokinase activate plasminogen indirectly by forming a complex with plasminogen, which subsequently behaves as a plasminogen activator to activate other plasminogen molecules by cleaving the arginyl-valine bond.
Plasmin is a different mechanistic class of thrombolytic agent that does not activate plasminogen. Plasmin directly cleaves fibrin in a thrombus, resulting in lysis. This avoids the requirement for plasminogen or plasminogen activators to be present in a thrombus. Many clots that are deficient in plasminogen due to thrombus contraction triggered by platelets and by Factor VIII.
Although tPA, SK, and UK have been successfully employed clinically to reduce a thrombotic occlusion, serious limitations persist with their use in current thrombolytic therapy. For example, because the systemic administration of tPA is not specifically targeted to the thrombus, it can result in significant systemic hemorrhage. Other limitations associated with plasminogen activators impact their overall usefulness. At best, the use of current thrombolytic therapy results in restored vascular blood flow within 90 minutes in only about 50% of patients, while acute coronary re-occlusion occurs in roughly 10% of the patients. Coronary recannulization requires on average 45 minutes or more, and intracerebral hemorrhage occurs in 0.3% to 0.7% of patients. Residual mortality is still about 50% of the mortality level in the absence of thrombolysis treatment.
A different approach that avoids many of the problems associated with the systemic administration of a plasminogen activator is to generate plasmin at the site of the thrombus or to directly administer the plasmin either into or proximally to the thrombus. Reich et al. in U.S. Pat. No. 5,288,489 discloses a fibrinolytic treatment that includes parenteral administration of plasmin into the body of a patient. The concentration and time of treatment were sufficient to allow active plasmin to attain a concentration at the site of an intravascular thrombus that is sufficient to lyse the thrombus or to reduce circulating fibrinogen levels. Reich et al. require generation of the plasmin from plasminogen immediately prior to its introduction into the body.
In contrast, Jenson in U.S. Pat. No. 3,950,513 discloses a porcine plasmin preparation that is asserted to be stabilized at low pH. However, such plasmin solution must be neutralized before systemic administration to humans for thrombolytic therapy.
Yago et al., in U.S. Pat. No. 5,879,923 discloses plasmin compositions employed as a diagnostic reagent. The compositions of Yago et al. consist low concentrations of plasmin at a neutral pH and an additional component that may be 1) an oligopeptide consisting of at least two amino acids, or 2) at least two amino acids, or 3) a single amino acid and a polyhydric alcohol, and the amino acids are specifically identified.
Numerous technical problems, such as the difficulty of preparing plasmin free of contaminating plasminogen activators, have prevented clinical use of plasmin. Plasmin preparations were typically extensively contaminated by the plasminogen activators streptokinase and urokinase, resulting in the attribution of thrombolytic activity to the contaminating plasminogen activators rather than to plasmin itself. The contaminating plasminogen activators can also trigger systemic bleeding at sites other than the targeted thrombosis. One factor limiting clinical use of plasmin is that plasmin, as a serine protease with broad specificity, is highly prone to autodegradation and loss of activity at physiological pH when prepared as a highly purified and highly concentrated solution. This provides severe challenges to the production of high-quality plasmin, to the stable formulation of this active protease for prolonged periods of storage prior to use, and to safe and localized administration of plasmin to human patients suffering from occlusive thrombi.
Thus, there is a need for a therapeutic composition comprising a stabilized serine protease capable of cleaving fibrin and a pharmaceutically acceptable carrier with a pH range sufficiently low to reversibly inactivate the serine protease, yet sufficiently high to limit acid hydrolysis of peptide bonds within the serine protease. Further, there is a need for such therapeutic composition to have a low buffer capacity to maintain low pH during storage, yet permit plasmin to rapidly revert to its active form at the pH in the local environment of the clot.
There is also a need for a therapeutic composition comprising a reversibly inactivated acidified serine protease stabilized by at least one pharmaceutically acceptable stabilizing agent and a pharmaceutically acceptable carrier.
These and other objectives and advantages of the invention will become fully apparent from the description and claims that follow or may be learned by the practice of the invention.